P818: DNP73 PROMOTES DRUG RESISTANCE AND IMMUNE EVASION IN MULTIPLE MYELOMA BY MODULATING MYC/CD47 AXIS
Authors: Lanting Liu, Jie Xu, Lixin Gong, Xiyue Sun, Lugui Qiu, Mu Hao
Journal: HemaSphere (2023)

Topic: 13. Myeloma and other monoclonal gammopathies - Biology & Translational Research Background: Multiple myeloma (MM) remains an incurable malignancy of plasma cells. The complex bone marrow microenvironment promotes resistance to both current anti-myeloma agents and emerging immunotherapies. Crosstalk between the NF-κB and p53 can play a pivotal role in various hematologic malignancies. DNp73, an inhibitor of the p53 tumor suppressor family, drives drug resistance and cancer progression in several solid malignancies. However, the biological functions and molecular mechanisms of DNp73 in MM remain unclear. In this study, we investigated the role of DNp73 in the drug resistance of MM, and disclosed the corresponding mechanism of how DNp73 promotes the immune escape of MM cells. Aims: To disclose the role of DNp73 in promoting drug resistance and immune evasion in MM, and explore the therapeutic strategy based on the mechanism research. Methods: We constructed DNp73 overexpression and sh-RNA interference plasmids to obtain stable cell lines. The effects of DNp73 on proliferation and drug sensitivity were determined by CCK8, flow cytometry and xenotransplantation model. To identify mechanisms of drug resistance, we here perform RNA-seq and CHIP-seq analyses in MM cell lines, while Western blot and RT-PCR were used to confirm the results. The DNA damage repair and invasion ability of MM cells were detected by immunofluorescence and transwell assay. To validate the role of DNp73 in immune escape, we also performed phagocytosis assays in vitro. Results: Our previous study reported that miR-15a was downregulated in MM cells and correlated with the inferior outcome of MM patients. Here, we demonstrated that downregulation the expression of miR-15a further increased the activation of NF-κB pathway (p<0.0001). Analysis of UCSC and ReMap experiment ChIP-seq data indicated that DNp73 is a direct target of p65. In addition, the level of miR-15a was negatively correlated with the expression of DNp73 in MM cells of patients (r=-0.672, p<0.05). DNp73 overexpression promoted the proliferation of MM cells, and induced the resistance to epirubicin treatment. After DNp73 was down-regulated, cell proliferation and drug resistance were effectively inhibited (p<0.001). Further in vivo study indicated the tumor volume in DNp73 knockdown group was significantly reduced, and increased sensitivity to the carfilzomib, epirubicin and pomalidomide was observed (p<0.01). Of note, RNA-seq analysis indicated that the levels of the transcription factors, MYC and NMYC, were highly correlated with the level of DNp73. GSEA analysis showed that MYC targets and DNA damage repair pathways were significantly enriched in DNp73-OE cells. Compared to control group, DNp73-OE cells were resistant to irradiation-induced cell death (p<0.01). Meanwhile, DNp73 knockdown significantly reduced the migration and invasion of cells in the transwell assay (p<0.01). CHIP-seq data showed that DNp73 could bind to the promoter region of NMYC. We also found that the expression of PD-L1 and CD47 were upregulated in the DNp73 high expression cells. DNp73 overexpression protects MM cells from phagocytosis, treated with anti-human CD47 antibody increased phagocytosis of macrophages (p<0.01). These analyses reveal a DNp73-governed protein resistance signature that includes cancer high-risk factors such as MYC and NMYC. Summary/Conclusion: In the present study, we demonstrated that miR-15a downregulation activated NF-κB, which promoted DNp73 expression at transcription level. DNp73 promotes drug resistance and immune evasion in multiple myeloma by modulating MYC/CD47 axis. This work identifies DNp73 is a potential target in MM treatment. Keywords: Multiple myeloma, Drug resistance, MYC